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Relationship of Endogenous Sex Hormones to Coronary Heart Disease: A Twin Study

Department of Medicine (K.H.M., R.M.), Stanford University School of Medicine, Stanford, California 94305; The Geriatrics Research, Education & Clinical Center (L.H., R.M.), Palo Alto Veterans Affairs Medical Center, Palo Alto, California 94304; Center for Health Sciences (R.E.K., H.J., G.E.S., D.C.), SRI International, Menlo Park, California 94025; and Department of Medical and Molecular Genetics (T.R.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Katharine H. Mikulec, M.D., Brigham and Women’s Hospital, Division of Endocrinology, Diabetes, and Hypertension, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: kmikulec{at}partners.org.

	Abstract

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We examined the association between endogenous sex hormones (estradiol, estrone, testosterone, and SHBG) and coronary heart disease (CHD) in white male twins. Stored plasma samples were available for 566 participants of the National Heart, Lung, and Blood Institute Twin Study, a longitudinal study of cardiovascular disease in male twins. Twenty-eight of these individuals were lost to follow-up, and outcome data were missing. Of the remaining 538 participants, 78 had CHD at baseline, and 154 subsequently developed CHD over 20 yr of follow-up. We observed no differences in mean unadjusted or age- and body mass index-adjusted log-transformed sex hormone concentrations for participants with and without CHD (all P > 0.08). Quartile and median split analyses revealed no significant association between any of the sex hormones and either prevalent or incident CHD. The discordant monozygotic twins showed no significant case-control group difference in estradiol, estrone, testosterone, and SHBG (all P > 0.3). The positive and negative concordant twin pairs had similar values for each of the sex hormones (all P > 0.3). We observed no relationship between endogenous sex hormone concentrations and prevalent or incident CHD in this sample of male twins.

	Introduction

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IT HAS BEEN recognized for many years that men are at higher risk for developing coronary heart disease (CHD) than women. The male-to-female ratio for fatal CHD is consistently around 2:1 in various countries with different lifestyles and rates of heart disease (1). The sex difference in CHD incidence persists after taking into account common CHD risk factors (2, 3, 4). CHD is rare among premenopausal women, and the Framingham Study revealed that the incidence rate of CHD in postmenopausal women is more than double that in premenopausal women of the same age (5, 6). These findings have led to the hypothesis that endogenous sex hormones may play a role in CHD in men and women. In particular, it has been postulated that estrogens provide protection against CHD; however, this remains to be proven and continues to be an area of controversy.

The Coronary Drug Project, a large multi-center trial initiated in 1965, examined whether exogenous estrogen prevented recurrent myocardial infarction (MI) in men who had already experienced one or more MIs. A 5.0-mg/d estrogen group was discontinued after an average of 18 months of treatment because of a higher incidence of MI. A 2.5-mg/d estrogen group was discontinued after 56 months because there was no change in incidence of MI, and there were trends toward an increased incidence of pulmonary embolism and thromboembolism (7, 8).

In addition, Phillips (9) observed signs of feminization and high endogenous estrogen concentrations among young men who had survived an MI. This finding prompted a number of investigators to study the association of endogenous sex hormone concentrations and CHD in men. In several case-control studies, cases with a prior history of MI were found to have elevated circulating concentrations of total estradiol and/or estrone (9, 10, 11, 12, 13, 14, 15, 16, 17). These findings led to the hypothesis that high estrogen exposure may actually increase the risk of CHD. However, these were hospital-based case-control studies, so causality could not be inferred. Two population-based case-control studies showed conflicting results with regard to estradiol and CHD in men; one showed increased concentrations among cases, and one showed no difference (18, 19). Neither study showed an association between testosterone and CHD.

No consistent association has been found between endogenous sex hormones and CHD in men diagnosed by angiography. Of six case-control studies that measured estradiol and/or testosterone, one observed high estradiol values and normal testosterone values in cases, two reported that cases had normal estradiol values and low testosterone values, and three found no significant between-group difference in either estradiol or testosterone (11, 12, 20, 21, 22, 23).

Of eight prospective studies (seven in men and one in men and women) in which baseline concentrations of one or more sex hormones and/or SHBG were examined in relation to the subsequent development of CHD, none showed a significant association (24, 25, 26, 27, 28, 29, 30, 31).

We had the opportunity to apply a unique analytic tool to this question using a large cohort of well-characterized male twins for whom stored plasma samples were available and the occurrence of CHD events was known. Studies of monozygotic (MZ) twin pairs are powerful because they eliminate many sources of variation (e.g. differences in genetic factors and in intrauterine and early environment influences) that are inherent in ordinary matched case-control studies of unrelated individuals. The results of this analysis are reported here.

	Subjects and Methods

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Study population

The National Heart, Lung, and Blood Institute Twin Study is a longitudinal study of cardiovascular disease in 514 pairs of male twins. The sample was drawn from a population-based registry of almost 16,000 pairs of white, male, Veteran twin pairs created and maintained by the Medical Follow-Up Agency of the National Academy of Sciences-National Research Council. The twins were born between 1917 and 1927 and were 43–56 yr of age when first examined between 1969 and 1973 at five research centers in the United States. Details of the study’s design and methods have been published elsewhere (32, 33). The protocol was approved by the Institutional Review Board, and all participants gave written informed consent. Four follow-up examinations have been completed (exam 2, 1981–1982; exam 3, 1986–1987; exam 4, 1995–1997; and exam 5, 1999–2000).

Data collection

Study protocol at each of the five examinations was similar. Attempts were made to see both members of each twin pair on the same day. A structured questionnaire was administered to determine health history and relevant lifestyle behaviors; height, weight, and systolic (SBP) and diastolic blood pressure were measured; and a 12-lead electrocardiogram was recorded. Blood samples were collected at the first three examinations after an overnight fast. Total, high-density lipoprotein (HDL) and low-density lipoprotein cholesterol and triglyceride concentrations were measured using the North American Lipid Research Clinics methodology. EDTA plasma was frozen at -70 C.

At each examination, subjects were interviewed by a physician who completed a medical history questionnaire that included questions about cardiovascular events and procedures. Medical records were obtained if subjects reported cardiovascular or cerebrovascular disease or if subjects reported hospitalizations or procedures relating to possible cardiovascular or cerebrovascular disease. At examinations 1, 2, and 3, two independent physicians reviewed the questionnaires and medical records. At examinations 4 and 5, one physician reviewed the questionnaires and medical records. Based on subject responses and review of medical records, physicians assigned corresponding International Classification of Diseases, Ninth Revision (ICD-9) codes. Death certificates or ICD-9 coding by the National Death Index were obtained for nearly all decedents. Death certificates were coded for underlying cause of death using ICD-9. In the present analysis, CHD is defined as ICD-9 codes 410–414. Subjects were considered lost to follow-up if they did not attend a subsequent examination and a death certificate or coding from the National Death Index was not available.

Between 2001 and 2002, estradiol, estrone, testosterone, and SHBG concentrations were measured in stored plasma samples from examinations 2 (1981–1982) or 3 (1986–1987). Specimens were from the same examination for both members of each twin pair. Variation in the amount of stored plasma available for hormone analysis accounted for missing data on a small number of participants, as shown in Table 1.

Hormone assays were carried out in the laboratory of one of the authors (R.M.) at the Palo Alto Veterans Affairs Heath Care System, as previously reported (34). Reagent kits were generously provided by Diagnostics Systems Laboratories, Inc. (Webster, TX). Attempts were made to run samples from both members of each twin pair in the same assay batch. Laboratory personnel were blinded to disease status and zygosity. Estradiol was measured by third-generation RIA, and the sensitivity and the intra- and interassay coefficients of variation were 0.6 pg/ml, 11.1%, and 14.1%, respectively. Estrone was measured by RIA, and the sensitivity and the intra- and interassay coefficients of variation were 1.2 pg/ml, 12.8%, and 11.3%, respectively. Total testosterone was measured by RIA, and the sensitivity and the intra- and interassay coefficients of variation were 0.08 ng/ml, 6.9%, and 10.8%, respectively. SHBG was measured by immunoradiometric assay, and the sensitivity and the intra- and interassay variation were 3 nmol/liter, 6.9%, and 7.0%, respectively.

Statistical analysis

The present analysis explores the association between sex hormone concentrations and CHD (ICD-9 codes 410–414) among subjects with follow-up, regardless of their CHD status at baseline. The SAS statistical package (SAS Institute, Cary, NC) was used for data analysis. Because of deviations from normality, logarithmic transformations of raw hormone concentration values were conducted. Hormone values greater than 3 SD from the logged mean were considered to be outliers and were not included in the present analysis.

We examined the relationship between hormone values and potential confounders using Pearson correlation analysis. A t test statistic was used to compare mean levels of established CHD risk factors between the CHD and non-CHD groups. The Mantel-Haenszel ² statistic was used to test for the association between treatment of hypertension and CHD status. An F test adjusted for the relationship of correlated observations was used to compare crude hormone mean levels between the CHD and non-CHD groups. A similar test was used to compare means adjusted for age and body mass index (BMI) between the CHD and non-CHD groups. We repeated these comparisons using PROC MIXED to account for the dependence among twins within pairs. The PROC RANK was used to identify quartile and median split membership. The association between quartile rank and CHD was examined for trend using the Mantel-Haenszel ² statistic. The relationship between median split and CHD was examined using a ² test. Multiple logistic regression analysis was performed for each sex hormone with the sex hormone as the independent variable, age and BMI as covariates, and CHD as the outcome variable. Baseline prevalence and incident CHD were first analyzed together and then analyzed separately. To estimate the concentrations of bioavailable estradiol and bioavailable testosterone, the ratio of total hormone to SHBG was calculated.

The analysis involving the MZ twins discordant for CHD (i.e. pairs in which one has CHD and the other sibling does not have CHD) and concordant for CHD (i.e. pairs in which both have CHD or both do not have CHD) was done in two steps. First, an F test that adjusted for the relationship of correlated observations was used to compare the means between the CHD and non-CHD members of the discordant twin pairs. This was followed by a comparison of the positive-concordant and negative-concordant twin pairs’ means via a similar F-test procedure. We repeated these comparisons using PROC MIXED with pair number being the group effect and individual twins within a pair representing repeated observations.

	Results

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Twin subjects analyzed as individuals

Hormone data were available for 566 subjects. Twenty-eight of these individuals were lost to follow-up, and outcome data were missing. Of the remaining 538 subjects, 250 came from 125 MZ twin pairs, 270 came from 135 dizygotic (DZ) twin pairs, and 18 were individuals for whom hormone data were not available for their twin brother. Baseline characteristics and mean hormone concentrations are shown in Table 1. The sex hormones were significantly associated with a group of established CHD risk factors (Table 2).

Table 3 shows mean unadjusted and age-BMI-adjusted log-transformed hormone concentrations for subjects with and without CHD. We observed no between-group differences in sex hormone values in the present sample (all P > 0.08). These results were confirmed after taking into account the dependence within twin pairs.

View larger version (44K): [in this window] [in a new window]   FIG. 1. Prevalence of CHD by quartiles of estradiol, estrone, testosterone, and SHBG (all P > 0.1).

Baseline prevalence and incident CHD events were analyzed separately. Quartile and median split analyses revealed no significant association between any of the sex hormones and either of the CHD outcomes.

Twin pair analyses

Table 4 shows the mean hormone concentrations for MZ twins who were discordant and concordant for CHD. The discordant MZ twins, representing genetically matched cases and controls, showed no significant case-control group difference in estradiol, estrone, testosterone, or SHBG (all P > 0.3). The positive and negative concordant twin pairs had similar values for estradiol, estrone, testosterone, and SHBG (all P > 0.3). We repeated the comparison of discordant pairs using PROC MIXED for MZ pairs and then again for MZ and DZ pairs combined. As expected, all P values were nonsignificant. We also repeated the comparison of positive and negative concordant twin pairs using PROC MIXED for MZ pairs and then again for MZ and DZ pairs combined. As expected, all P values were nonsignificant.

	Discussion

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First, MZ twins discordant for CHD represent cases and controls matched for genetics, intrauterine environment, and early environment. This model eliminates many sources of variation that are inherent in ordinary case-control studies, most notably genetic make-up.

Second, most of the previously published case-control studies in men were hospital-based, and in several of these studies, blood samples were drawn within days of acute MI (11, 13, 14, 15, 16). By contrast, the subjects in our study were ambulatory without evidence of acute illness at the time of phlebotomy. Elevated estrogen concentrations in the setting of acute MI may be related to general stress rather than to CHD. In men, 95% of daily estrone production is derived from aromatization of the adrenal androgen, androstenedione, and as such, it is highly responsive to adrenal stimulation by ACTH (35, 36). Estrone production is increased in intensive care unit patients who do not have apparent CHD (13), and estrone and urinary catecholamines are significantly correlated in acutely ill subjects (16). Estradiol, by contrast, does not appear to be under the direct control of ACTH (35, 36, 37), although some estradiol is produced from circulating estrone (38). It is also likely that medications administered during hospitalization for acute MI may influence estradiol measurements. Thus, general stress and medications may have confounded the sex hormone measurements of hospital-based case-control studies.

There are several important limitations of the current twin study. For each subject, hormone measurements were made on a single plasma sample. Because steroid hormone concentrations undergo substantial daily fluctuation, a single hormone measurement may not suffice to accurately describe an individual’s overall hormone status. Thus, multiple specimens may be needed to assess the overall hormone status of an individual (2, 24). However, the impact of such fluctuation is minimized in this study by the large number of subjects.

A second methodological issue in our study is sample deterioration resulting from the storage of plasma samples for 15–22 yr before hormone measurement. It has been suggested that estradiol and estrone values decrease with time in storage, even at -70 C (24, 26, 39). We note, however, that the values we report for estradiol, testosterone, and SHBG fall within expected ranges for normal men, although for estrone, the values are slightly low.

Total estradiol and total testosterone were determined, but total measurements may not estimate hormone exposure as well as measurements of bioavailable or free hormone. Attempts were made to approximate bioavailable estradiol and testosterone by calculating the ratios between the total hormones and SHBG. Such analyses also revealed no significant associations with CHD, but we cannot exclude the possibility that such relationships would have emerged if we had directly assessed bioavailable or free hormone concentrations.

Many of the prior studies investigating sex hormones and CHD share these same potential shortcomings. There is no reason to believe, however, that male individuals with or without CHD would be preferentially vulnerable to any of these potential confounders. It is most likely that all subjects would be similarly affected and that valid relationships could still emerge despite these issues.

Although the results of this study do not confirm a relationship between sex hormone status and CHD, multiple lines of evidence support the hypothesis that there should be an association. High-affinity estradiol receptors are present in both vascular smooth muscle and endothelium (40, 41). We know that estrogens inhibit smooth muscle proliferation (42) and decrease smooth muscle tone (43). Estrogens enhance the release of nitric oxide and prostacyclin from endothelial cells, thus inducing vasodilation (44, 45, 46). Furthermore, estrogens exert indirect effects on the cardiovascular system through their influence on lipoprotein metabolism and the coagulation, fibrinolytic, and antioxidant systems (47). Thus, plausible mechanisms exist for an estrogen effect on CHD. In addition, a 31-yr-old man with estrogen unresponsiveness due to a mutation in the estrogen receptor- was reported to have early coronary calcifications (48) and peripheral endothelial dysfunction as shown by absence of flow-mediated vasodilation in the brachial artery (49), thus providing additional evidence for a role of estrogen in cardiovascular health in men.

It may be that hormone concentrations per se do not portray an integrated view of hormone action. Other variables, such as coactivators, corepressors, hormone metabolism, receptor polymorphism, and receptor number make important contributions to hormonal status (44, 47). In addition, the pattern of hormone exposure may also be important. In premenopausal women, estradiol concentrations range from 40–200 pg/ml in the follicular phase and from 250–500 pg/ml at the time of ovulation (44). Several hormones, such as gonadotropin-releasing hormone (50) and PTH (51, 52), are known to exert dramatically different influences when presented to their target tissues in an intermittent or continuous fashion. Perhaps a similar phenomenon occurs with sex hormones, and this may be a fruitful area for additional research.

	Footnotes

 
Supported in part by the National Heart, Lung, and Blood Institute Grant R01-HL51429 and by the Department of Veteran Affairs.

Current address for K.H.M.: Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115.

Current address for R.M.: Eli Lilly & Company, Indianapolis, Indiana 46285.

Abbreviations: BMI, Body mass index; CHD, coronary heart disease; DZ, dizygotic; HDL, high-density lipoprotein; ICD-9, International Classification of Diseases, Ninth Revision; MI, myocardial infarction; MZ, monozygotic; SBP, systolic blood pressure.

Received July 31, 2003.

Accepted November 24, 2003.

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